Protein in which electrical interaction is introduced within hydrophobic interaction site and preparation method therefor

ABSTRACT

The present invention provides a protein or antibody in which, of a pair of hydrophobic amino acids selected from within a hydrophobic interaction site of the protein, one hydrophobic amino acid is transformed into a substance having a positive electrical charge and the other hydrophobic amino acid is transformed into a substance having a negative electrical charge, and electrostatic interaction is introduced within the hydrophobic interaction site of the protein by means of the positive charge and the negative charge. The present invention also provides a method for preparing the protein or antibody, and a method for measuring the degree of coupling between a heavy chain and a light chain, using the antibody. The protein or antibody in accordance with to the present invention has a low contamination by a homodimer or a monomer, and thus a heterodimer can be obtained in high purity.

TECHNICAL FIELD

The present invention relates to heterologous bispecific antibodies(BsAbs) or bispecific fusion proteins (BsFps) with high purity.

BACKGROUND ART

Most of bispecific antibodies (BsAbs) are artificially manufactured tobind two different targets simultaneously rather than generally producedin nature. A double targeting ability provides BsAbs with new applicablefield, which has not been managed by monopecific antibodies (MsAbs).Special interest in therapeutic purposes is the provoking possibility,such that BsAbs (1) reliably recruit immune cells into the proximity oftarget cells, (2) inhibit or activate two distantly apart signalingpathways in target cells to create synergetic effects, and (3) deliverradiation-induced therapeutic substances, medical drugs, toxins orsignaling molecules in a specific- and regulatory manner.

BsAbs are generally utilized for delivering T cells to tumor cells in aMHC-independent way, mediating a linkage between cell surface antigensof tumor cells and CD3-TCR complex of cytotoxic T cells (FIG. 1).Catumaxomab(Removab®), rat-mouse hybrid monoclonal antibody, in FIG. 1is used to treat malignant ascites, which is called ‘Trifunctionalantibody.”

Complete chain association should occur at two different levels, inorder to produce minimally modified full-length IgG-like BsAbs withoutany chain association problem. (1) Two heavy chains should beheterologous bispecific, and (2) two light chains (LC) should paircorrectly with their respective heavy chains.

Chain association issues should be solved to produce BsAbs in atrustworthy method. As shown in FIG. 2, combination of two heavy chainsand two light chains generates 10 different forms of antibody chimera.Among them, only one is a correct BsAb, and the rest are worthlessChimera. This chain association issue reduces production yield ofcorrect BsAb to at least 10 times in industry fields, and causes variousproblems with difficulties in isolating BsAbs from other chimera.Therefore, many pharmaceutical companies spend a lot of resources andmake efforts to develop and obtain technology for producing BsAbs in adirect and reliable way.

Many various BsAb-related techniques (45 different formats) have beendeveloped. These techniques are classified into 4 categories based onthe structure. First, heterologous bispecification of heavy chains byvarious methods comprising structural complementarity kown toKnob-into-Hole or simply KiH, electrostatic steering effect, or CH3domain shuffling (called to SEEDbody™); second, various antibodyfragment formats such as Diabody™, BiTE™ and DART™; third, technologyusing one or more functional domains combined with intact antibodies,such as Modular Antibody™, Zybody™, dAbs™ and DVD-IG™; and fourth,techniques adopting full length IgG-like scheme as Duobody™ (Fab-ArmExchange), CrossMab™, Azymetric™, and kI body™ have been developed.

Out of them, Zymeworks through the United States Patent Application No.2013-892198, claiming a patent for the structure of heteromultimerimmunoglobulin chains having mutations in Fc domain, showed that theantibodies of the heterologous multimeric structure could be made bymodifying cysteine residues involved in disulfide bonds with chargedamino acids.

However, any patent above has not disclosed such a technology that amodified amino acid pair selected from the portion of the hydrophobicinteraction induces to selectively couple each other by theelectrostatic interaction. The inventors have completed the presentinvention by confirming that heterologous bispecification takes placemore selectively when one pair of amino acids involved in hydrophobicinteraction are modified to an acidic amino acid and a basic amino acid,respectively.

SUMMARY OF THE INVENTION Technical Problem

The present invention aims to provide bispecific antibodies withexcellent heterologous bispecification (heterodimer).

The other purpose of the present invention is to provide a method formanufacturing proteins that heterologous bispecifications occur well byaltering a pair of amino acids in the hydrophobic interactions to thecharge opposite to each other

Technical Solution

For the above object, the first aspect of the present invention is toprovide proteins, which the electrostatic interaction has beenintroduced by the above negative charge and the above positive charge,in that a pair of hydrophobic amino acids selected from the portion ofthe hydrophobic interaction of the protein, altering one hydrophobicamino acid to a positive charge, and the other hydrophobic amino acid tonegative charge. Materials having the positive charge may be basic aminoacids may be but not limited to the same, materials having the negativecharge may be acidic amino acids but not limited to the same.

The hydrophobic amino acid is any of the amino acid selected from agroup consisting of glycine, alanine, valine, leucine, isoleucine,methionine, proline, and phenylalanine, and the acidic amino acid is anyone of amino acid selected from a group of aspartic acid or glutamicacid.

Assembly of full-length IgG-like bispecific antibodies from twodifferent HC/LC pairs is made from two chain association processes. Inother words, HC heterologous bispecification and productive HC/LCpairing, and their success rates between the two heavy chains, depend onthe efficiency of distinguishing between the heavy chain and the lightchain.

To find the appropriate variation site, amino acid residues on thehydrophobic interface between the chains of the antibody have beenfocused since the hydrophobic interaction is the main driving force forfolding and binding of the protein. For selecting an appropriate type ofmodifications as powerful as a hydrophobic interaction, since itprovides the discernibility of the protein necessary to solve chainassociation problem of bispecific antibodies, the electrostaticinteraction has been chosen.

Distinction between such chains was conceived to be solved byintroducing the complementary pairing of structural modifications at theinterface between the two binding chains. One or more hydrophobic aminoacids were replaced by mutated charged amino acid to pair with thecounterpart. Such a change is hereafter called SHOCAP (substitution ofhydrophobic into oppositely charged amino acid pair). SHOCAP in the Fcdomain of two the heavy chains generates the positively and negativelycharged heavy chains (each called Ha and Hb). These electrostaticinteractions prefer a heterologous bispecification (heterodimerization)than homologous bispecification (homodimerization) of the heavy chain.In the same way, modifications of two Fab domains by SHOCAP createpositively and negatively charged light chains (called La and Lb,respectively), and the electrostatic interactions with the oppositelycharged Fab domains of the heavy chains increase the probability ofcorrect HC/LC pairing.

To produce BsAbs without any chain association problem, two independentmodifications were introduced into naturally-occurring antibodies. Oneis for heterologous bispecification of the Fc domain and the other forthe correct pairing of the heavy chain and the light chain.

In the present invention, heterologous bispecification(heterodimerization) of the Fc domain occurs firmly and actively if oneor more hydrophobic interactions in the Fc domain of the antibody areconverted to electrostatic pair interactions (for example, conversion ofthe hydrophobic interaction of two tyrosines at the 407th residues(Y407: Y407) to the electrostatic pair interaction of aspartate andlysine the 407th residues (D407: K407)).

The use of the SHOCAP technology can easily distinguish two heavy chains(heavy chain) and two light chains (light chain) when major hydrophobicresidues of the Fab domain was substituted (for example, leucine of the128th residue and phenylalanine of the 118th residue (L128: F118) tolysine of the 128th residue and aspartic acid of the 118th residue(K128: D118). Therefore, IgG-like bispecific antibodies can be generatedby applying SHOCAP technology to the Fc and Fab domains of antibodies.

The second aspect of the present invention is to provide antibodies inwhich the electrostatic interaction has been introduced into the regionof the hydrophobic interaction by mutating one hydrophobic amino acid toa positively charged amino acid and the other to a negative charge. Theabove positive charge may be basic but not limited to the same and theabove negative charge may be acidic but not limited to the same.

Specifically, antibodies have binding forces by electrostaticinteractions between any one of the amino acid mutated to an acidicamino acid selected from aspartic acid or glutamic acid, and the otherto a basic amino acid selected from lysine, arginine, or histidine, ofwhich mutations were introduced to one or more amino acid pairs selectedfrom the group of amino acid pairs, consisting of 351 leucine and 351leucine pair, 395 proline and 397 valine pair, 395 proline and 395proline pair, 407 tyrosine and 407 tyrosine pair of hydrophobicinteraction in CH3 domain; and 128 leucine and 118 phenylalanine pair,128 leucine and 133 valine pair, 141 alanine and 116 phenylalanine pair,141 alanine and 135 leucine pair, 145 leucine and 133 valine pair, 170phenylalanine and 135 leucine pair, 185 valine and 118 phenylalaninepair and 185 valine and 135 leucine pair between CH1 domain and CLdomain of human antibody. More specifically, antibodies arecharacterized to be mutated on a set of combination selected from thegroup consisting of Z0 to Z14 in Table 4. The antibodies can have a pairof ectodomains with a pair of functions. The ectodomain can play a rolein cancer and signaling, and this toxin may be used to treat cancercoupling cell death bound by another specific antibody. The ectodomainsmay be a pair of ectodomains selected from the group consisting of TNR2,Her3, Tie2, TGFbR1, BMPbR1, Il-12R-b1, IL-4Ra, ITGA4, ITGA2B, INFAR1,IL-12A, IL-4, InFa, BMP2, IL-1R1L, IL-17RA, IL-17A, Fas, FltD2, Her1,Tie1, TGFbR2, IL-12R-b2, IL-13Ra1, ITGB1, ITGB3, INFAR2, IL-12B, IL-13,INFb, BMP7, IL-1RAP , IL-17RC, and IL-17F, but not limited to the same.More specifically, the antibodies may be fused with a combination ofectodomains selected from a combination group consisting of Her2/FltD2combination, Her1/Her3 combination, and Tie/Tie2 Ectoin. Morespecifically, it may be bispecific antibodies having the heavy chainmutated to Z14 combination and the light chain simultaneously including4D9 ectodomain specific for A-type influenza virus and 2B9 ectodomainspecific for B-type influenza virus. More specifically, the aboveantibodies may be the antibodies with enhanced pairing between the heavyand the light chains, consisting of any combination selected from acombination group consisting of V1 to V5, W1 to W8, V2p, V3p, W4p, V3W4,W4v3, and V3v1 of one heavy chain (HP) and the other the heavy chain(HN), and one light chain (LP) and the other light chain (LN) shown inTable 7. More specifically, the above antibodies may be the antibodieswith enhanced pairing between the heavy chain and the light chain,consisting of tryptophan-to-lysine mutant at the 103th residue,lysine-to-aspartic acid mutant at the 128th residue,phenylalanine-to-lysine mutant at the 118th residue, proline-to-asparticacid mutant at the 44th residue.

The third aspect of the present invention is to provide the method ofmanufacturing proteins of which chain selectivity has been increased,comprising of the steps such as;

-   -   (1) selecting a pair of hydrophobic amino acids in the region of        the hydrophobic interaction between the polypeptide chain and        the polypeptide chain;    -   (2) modifying one hydrophobic amino acid to a positive charge,        and the other hydrophobic amino acid to a negative charge in the        selected pair of amino acids; and    -   (3) binding by the electrostatic interactions through contacting        between the positive and negative charges. Material having the        positive charge may be a basic amino acid but not limited to the        same, and material having the negative charge may be an acidic        amino acid but not limited to the same.

A fourth aspect of the present invention is to provide a method formeasuring the extent of pairing between the heavy chain and the lightchain of BsAbs using BsAbs having the heavy chain mutated to the Z14combination and the common light chain including 4D9 ectodomain specificfor A-type influenza virus and 2B9 ectodomain specific for B-typeinfluenza virus.

Advantageous Effects

SHOCAP modifications of the heavy chain of the Fc domain create thepositively and negatively charged heavy chain (called Ha and Hb eachhereinafter). These electrostatic interactions prefer heterologousbispecification (heterodimerization) than homologous bispecification(homodimerization) of the heavy chain. In the same way, SHOCAPmodifications of two Fab domains generate the positively and negativelycharged light chains (called La and Lb, respectively). And theelectrostatic interaction of oppositely charged Fab domain of the heavychain increases the probability of correct HC/LC pairing. Accordingly,the antibodies in the present invention can obtain heterologousbispecific antibodies or proteins which are less contaminated withhomodimers or monomers.

Another advantage of the present invention is to induce less immunerejection since mutations on minimal number of amino acids have notcaused any significant structural changes of natural antibodies, andfurther target residues have been buried deep in the surface ofhydrophobic interaction between the heavy and light chains.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a general form of Bispecific Antibody. As an example,Catumaxomab (Removab®) rat-mouse hybrid monoclonal antibody is used totreat malignant ascites.

FIG. 2 shows that a combination of two light chains and two heavy chainsgenerates 10 different antibody chimera.

FIG. 3 shows amino acids which are involved in hydrophobic interactionsof antibodies.

FIG. 4 shows that the amino acid sequences are highly conserved in theFc domains of antibodies between human and mouse.

FIG. 5 shows14 sets of mutations in the Fc portion bound by respectiveTNFR2 ectodomain and FAS ectodomain, which introduced the electrostaticinteractions, in order to show the efficiency of heterologousbispecification. Positively charged amino acids were inserted into theregions of hydrophobic interactions in chain A and, negatively chargedamino acids was inserted into the regions of hydrophobic interactions inchain B.

FIG. 6 shows how well heterologous bispecifications of Fc occur in theset of Z0 to Z4 in Table 4 by SDS-PAGE analysis.

FIG. 7 shows how well heterologous bispecifications of Fc occur in theset of Z5 to Z9 in Table 4 by SDS-PAGE analysis.

FIG. 8 shows how well heterologous bispecifications of Fc occur in theset of Z10 to Z14 in Table 4 by SDS-PAGE analysis.

FIG. 9 is SDS-PAGE analysis of comparing the efficiency of heterologousbispecifications between heterologous Her3/FltD2 BsAbs and controlantibodies which are generated based on the Z14. A) schematic diagram ofa heterologous BsAb, B) SDS-PAGE analysis

FIG. 10 compares the efficiency of heterologous bispecifications betweenheterologous Her1/Her3 BsAb and control antibody which are generatedbased on the Z14. (A) schematic diagram of a heterologous BsAb,Her1/Her3, (B) SDS-PAGE analysis, (C) HIC-HPLC analysis

FIG. 11 compares the efficiency of heterologous bispecifications betweenheterologous Tie1/Tie2 BsAb and control antibody which are generatedbased on the Z14. (A) schematic diagram of a heterologous BsAb,Tie1/Tie2, (B) SDS-PAGE analysis, (C) HIC-HPLC analysis

FIG. 12 shows heterologous bispecification to share a common light chainof the antibody. BsAbs are only made possible by the heterologousbispecification of Fc because it does not need correct pairing of theheavy chain and light chains.

FIG. 13 is a schematic diagram showing that 3 types of antibodies, butnot the 10 types, are made in the case of antibodies sharing a commonlight chain

FIG. 14 shows that, from a view as a result of the result of SDS-PAGEanalysis, BsAbs are purely produced in the case of antibodies of sharingthe common light chains prepared based on the Z14 in Table 4.

FIG. 15 shows that, from a view as a result of the result of HIS-HPLCchromatography, BsAbs are purely produced in the case of antibodies ofsharing the common light chains prepared based on the Z14 in Table 4.

FIG. 16 shows that the antibodies produced based on the Z14 in Table 4have bispecific antigen-binding activity. 4D9 (HA0+LC) and 2B9 (HB)+LC)were used as control.

FIG. 17 shows that the Fc of antibodies generated in the presentinvention binds well with receptors.

FIG. 18 shows pharmacokinetic data of BsAbs having the common lightchain manufactured by the present invention.

FIG. 19 is a schematic diagram showing that, in the manufacture ofantibodies in accordance with the present invention, binding of theheavy and light chains is distinguished to be symmetrical orasymmetrical relative to the positions of the charge.

FIG. 20 shows that pairing of the heavy chains and the light chains ofthe antibodies in accordance with the present invention is correct. Thecombination of V3W1 in Table 7 is the most excellent pair between theheavy chain and the light chain.

BEST MODE

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not to be construedto limit the scope of the present invention.

EXAMPLE 1 Heterodimerization of the Fc Domain A) Selection ofModification Sites and Types

To search for the appropriate sites of SHOCAP modification in both Fcand Fa, hydrophobic contacts between the antibody chains were analyzedby Protein Interaction Calculator (PIC). A number of hydrophobicinteractions between the chains have been found throughout the entireantibody domains. Five pairs of residues in two CH3 domains wereinvolved in the hydrophobic interactions between the chains (see Table 1and FIG. 3). Three hydrophobic interactions at different sites in thetwo CH3 domains were distributed symmetrically. One region is made ofmutual hydrophobic interactions between P395 and V397, and the other tworegions are made of the interaction of a hydrophobic pair of Y407 andL351, respectively. These residues were found to be highly conservedbetween human and mouse (and also other mammals) antibody classes (FIG.4), indicating that these hydrophobic pair interactions might be pivotalin maintaining the dimeric structural integrity of the Fc domains. Ninepairs of residues are involved in the hydrophobic interactions betweenCH1 and CL (Table 2, FIG. 3). Except in the case of residues in the CDR,a total of 12 pairs of residues from the VH and VL domains were involvedin hydrophobic interactions between the chains (see Table 3 and FIG. 3).No notable interaction exists in the interface between CH2-CH2 domain.

TABLE 1 HIP No. CH3 Domain (Chain A) CH3 Domain (Chain B) 1 L351 L351 2P395 V397 3 P395 P395 4 V397 P395 5 Y407 Y407

TABLE 2 HIP No. CH1 Domain CL Domain 6 L128 F118 7 L128 V133 8 A141 F1169 A141 F118 10 A141 L135 11 L145 V133 12 F170 L135 13 V185 F118 14 V185L135

TABLE 3 HIP No. VH Domain VL Domain 15 V37 F98 16 F45 P44 17 F45 Y87 18F45 F98 19 W47 Y96 20 W47 F98 21 W50 Y96 22 Y91 P44 23 W103 Y36 24 W103Y36 25 W103 A43 26 W103 P44 27 W103 F98

To solve problems with chain association in the heterologousbispecification of Fc, five pairs of residues consisting of four otherhydrophobic residues (L351, P395, V397 and V407) that span the two CH3domains have been considered as major modification sites.

The four hydrophobic residues (L351, P393, V397 and Y407) aretransformed, each or in combination, into electrostatic interactionpairs to produce a total of 14 sets of TNFR2-Fc and FAS-Fc variants(Table 4 and FIG. 5).

TABLE 4 Set No. Chain A (TNFR2) Chain B (FAS) Z0 A0 — B0 — Z1 A1 L351KB1 L351D Z2 A2 P395K B2 P395D Z3 A3 Y407K B3 Y407D Z4 A4 L351K/P395K B4L351D/P395D Z5 A5 L351K/Y407K B5 L351D/Y407D Z6 A6 T394K/P395K B6T394D/P395D Z7 A7 T394K/V397K B7 T394D/V397D Z8 A8 P395K/V397K B8P395D/V397D Z9 A9 P395K/Y407K B9 P395D/Y407D Z10 1A10 L351K/T394K/P395KB10 L351D/T394D/P395D Z11 1A11 L351K/T394K/V397K B11 L351D/T394D/V397DZ12 1A12 L351K/P395K/V397K B12 L351D/P395D/V397D Z13 1A13L351K/P395K/Y407K B13 L351D/P395D/Y407D Z14 1A14 L351K/T394K/P395K/ B14L351D/T394D/P395D/ V397K V397D

The potential of heterologous bispecification of those 14 sets of themutants was examined using SDS-PAGE analysis. In order to facilitate theinterpretation of the results, two receptors having ectodomains withdifferent molecular size and distinct ligand-binding activity wereselected. The hydrophobic moiety in these large Fc ectodomains of TNFR2capable of binding to TNFα was mutated to the positively charged moiety(called “A chain” hereinafter). The hydrophobic moiety in these small Fcectodomains of FAS having a binding affinity for FASL was substituted tothe negatively charged moiety (called “B chain” hereinafter).

In each of the variants set, A and B chains were independently expressedat the same time. Fc fusion proteins produced from sets of single (A andB) and coexpression (A+B) have been purified by protein Achromatography. Proteins were finally eluted with 1 ml of protein Aelution buffer solution, 10 μl out of eluted protein fractions wereanalyzed by 10% SDS-PAGE. The possibility of the heterologousbispecification was determined by comparing the band density ofheterologous bispecific TNFR2-Fc/FAS-Fc with that of homologousbispecific (TNFR2-Fc)₂ and (FAS-Fc)₂ in the coexpression setting (A+B).In addition, monomeric TNFR2-Fc and FAS-Fc products were compared withhomologous bispecific (TNFR2-Fc)₂ and (FAS-Fc)₂ products in the set ofsingle expression (A and B). FIG. 7 shows the data set of Z5-Z9variants. FIG. 8 shows the data set of Z10-Z14 variants. A set of Z14mutant was finally selected as the best set. The probability of theheterologous bispecification of Z14 variant was likely better than anyothers. In this set, heterologous bisepcific TNFR2-Fc/FAS-Fc was themost excellent compared to homologous bispecific (TNFR2-Fc)2 and(FAS-Fc)₂ in the set of coexpression, and monomeric TNFR2-Fc and FAS-Fcproducts were the most unstable in the set of single expression. Itshows that the antibodies in accordance with the present invention areless contaminated with monomeric or homologous bispecific variants.

EXAMPLE 2 Preparation of Heterologous BsAbs Based on Z14 in Table 4

By employing heterologous bispecification of Fc domains based on Z14, atotal of 17 different heterologous bispecific Fc fusion proteins(BsFcFs) for various target diseases in Table 5.

TABLE 5 Fc Origin A Chain B Chain Target Diseases I Human (G1) TNFR2 FasAutoimmune Diseases II Human (G1) Her3 Flt1D2 Cancer III Human (G1) Her3Her1 Cancer IV Human (G1) Tie2 Tie1 Cancer V Human (G1) TGFbR1 TGFbR2Fibrosis, Wound healing and Cancer VI Human (G1) BMPbR1 BMPbR2Osteopetrosis VII Human (G1) IL-12R-b1 IL-12R-b2 Autoimmune Diseases(Psoriasis, MS and Crohn) VIII Human (G1) IL-4Ra IL-13Ra1 Asthma andAtopy IX Human (G1) ITGA4 ITGB1 MS X Human (G1) ITGA2B ITGB3 ThrombosisXI Human (G1) INFAR1 INFAR2 Autoimmune Diseases XII Human (G2) IL-12AIL-12B Immunotherapeutic anticancer agent XIII Human (G2) IL-4 IL-13Autoimmune Diseases (Psoriasis, MS and Crohn) XIV Human (G2) INFa INFbCander, Hepatocarcinoma, MS XV Human (u/dG2) BMP2 BMP7 Osteopetrosis XVIHuman (E) IL-1R1L IL-1RAP Atopy (anti IL-33 blocker) XVIII Human (G1)IL-17RA IL-17RC Autoimmune Diseases (Psoriasis, MS and Crohn) XIV Human(u/dG2) IL-17A IL-17F Immunotherapeutic anticancer agent

A. Design for Heterologous Bispecific Her3/FltD2 Based on Z14

Her3 ectodomain was fused to the positively charged Fc domain (A chain;97 kD), and F1tD2 domain was to the negatively charged (B chain, 40 kD).Positive extracellular domains have been reported to retain thepossibility of inherent homologous bispecification.

SDS-PAGE Patterns

Two matched chains were co-expressed, purified by protein Achromatography, and analyzed by 10% SDS-PAGE. Z14 heterologousbispecification was superior to others since monomeric Her3-Fc andFltD2-Fc were seen in the three different control sets but not in Z14.

B. Design for Heterologous Bispecific Her1/Her3 Based on Z14

Her3 ectodomain was fused to the positively charged Fc domain (A chain;97 kD), and Her1 domain was to the negatively charged (B chain, 95 kD).Positive extracellular domains have been known to maintain thepossibility of inherent homologous bispecification.

(1) SDS-PAGE Pattern

Two matched chains were co-expressed, purified by protein Achromatography, and analyzed by 10% SDS-PAGE. The possibility of Z14heterologous bispecification was high enough to overcome inherenthomologous bispecification of Her1 and Her3 although monomeric Her3-Fcand Her1-Fc forms were seen in Z0 set (FIG. 10 (B)).

(2) HIC-HPLC Analysis

20 μl of the concentrated sample (1˜5mg/ml) was loaded onto TSK gelphenyl HIC column. Linear gradient from 60 to 100% acetonitrile wasapplied with a flow rate of 0.1 ml/min for 40 minutes. Observation wascarried out at 214 and 280 nm. Similar to the SDS-PAGE pattern, Z14showed a high potential for heterologous bispecification (see FIG. 10(C)).

C. Design for Heterologous Bispecific Tie/Tie2 Based on Z14

Tie1 ectodomain was fused to the positively charged Fc domain (A chain),and Tie2 domain was to the negatively charged (B chain).

(1) SDS-PAGE Pattern

Two matched chains were co-expressed, purified by protein Achromatography, and analyzed by 10% SDS-PAGE. Monomeric Tie1-Fc andTie2-Fc forms were seen in Z0 set but not in Z14. It indicates that Z14retains the excellent potential for heterologous bispecification (seeFIG. 11 (B)).

(2) HIC-HPLC Analysis

The HIC-HPLC analysis was performed as described previously. Similar tothe SDS-PAGE pattern, it indicates that a potential for heterologousbispecification of Z14 is high (see FIG. 11 (C)).

EXAMPLE 3 Preparation of Heterologous BsAbs from Antibodies havingCommon Light Chains

Two fully humanized antibodies have been found by the technique of phagedisplay. One is 4D9, specific for A-type influenza virus, and the otheris 2B9, specific for B-type influenza virus. Interestingly, it has beenfound that the two antibodies share a single common light chain (seeFIG. 12). A common light chain bispecific antibody (CLC-BsAb) wasdesigned using the benefits of sharing the common light chain in the twoantibodies. Dual specific antibodies may be formed only by Fcheterologous bispecification (that is, the process of correct pairing ofHC/LC can be omitted).

When natural two heavy pairs with a common light chain, three (not 10)possible antibodies are produced (see FIG. 13). Three control sets andZ14 set were made as shown in Table 6.

TABLE 6 Set HA HB LC Z0 HA0 — HB0 — — Z14 HA14 L351K/T394K/ HB14L351D/T394D/ — P395K/V397K P395D/V397D CPC HPC D399K/E356K HNCK409D/K392D KiH HKC T366S/L368A/Y407V HHC T366W AzS HAC T350V/T366L/ HBCT350V/L351Y/ K392L/T394W F405A/Y407V

(1) Analysis of Result

As expected, a natural set (Z0) generated the three possible antibodiesin SDS-PAGE analysis. (HB/LC)₂, (HA/LC)//(HB/LC) and (HA/LC)₂. Incontrast, Z14 set only produced bispecific forms (HA/LC)//(HB/LC) (seeFIG. 14). Z14 set did not produce monomeric antibodies at any visiblelevels. It reveals that the samples co-expresed by Z14 set is notcontaminated with monomeric antibodies and is highly pure with onlybispecific antibody forms.

(2) HIC Analysis Purity of Z14 was evaluated by HIC-HPLC chromatography.Z0 set has three peaks corresponding to each (HA/LC)₂, (HA/LC)//(HB/LC)and (HB/LC)₂. In contrast, Z14 set has a single peak corresponding tobispecific (HA/LC)//(HB/LC) antibodies. It reveals that, similar to theSDS-PAGE result, the samples co-expresed by Z14 set is not contaminatedwith monomeric antibodies and is highly pure with only bispecificantibody forms (see FIG. 15).

(3) Confirmation of Dual Antigen-Binding Activity

Two antigens, A-HA5 and B-HA, were separately coated in three differentamounts (100, 50 and 25 ng/well) on the ELISA plate. After blocking withblocking solution, 100 ng of the antibody was added and incubatedovernight. The plate was thoroughly washed, and HRP-conjugatedanti-human Fc mouse antibody was added to each well.

Result Analysis

Z14 has dual binding activity against both A-HA5 and B-HA antigens. Onthe other hand, the two control antibodies of 4D9 (HA0+LC) and 2B((HB0+LC) showed single binding activity (see FIG. 16).

EXAMPLE 4 Measurement of the Receptor Binding Activity of the Fc Domainof Antibodies

100 ng of FcRn-Fc fusion proteins were coated in each well of the ELISAplate. After blocking with blocking solution, 100 ng of 4D9 (HA0+LC),2B9 (HB0+LC), and Z14 antibodies were added and incubated overnight. Theplate was thoroughly washed, and HRP-conjugated anti-human Fc mouseantibody was added to each well.

Result analysis showed that the receptor binding activities of the Fcdomains of 4D9 (HA0+LC), 2B9 (HB0+LC), and Z14 antibodies were notsignificantly different (FIG. 17).

EXAMPLE 5 Pharmacokinetic Analysis of Bispecific Antibodies Manufacturedin the Present Invention

10 mg/ml of Remicade and 4 mg/kg of B6CBA were injected into miceCLC-BsAb, sample concentrations were measured up to 14 days afteradministration. There was no significant difference in the profile ofthe concentrations between the two antibodies. In order to analyze thepharmacokinetic parameters between Remicade and CLC-BsAbs, ANOVA testwas performed with several pharmacokinetic parameters (CL, V1, AUC, MRT,t1/2), which was calculated by analysis of NCA and two-compartment model(see FIG. 18).

EXAMPLE 6 Correct Pairing of the Heavy Chains (HC) and the Light Chains(LC) of Antibodies Manufactured in the Present Invention

To solve the problem at the step of pairing of HC/LC chains, 12hydrophobic residues (Y36, A43, P44, Y87, Y96 and F98 of VL domain, andV37, F45, W47, W50, Y91, and W103 of the VH domain) and 9 hydrophobic(F116, F118, L133 and L135 within the CL domain, and L128, A141, L145,F170 and V185 of the CH1 domain) have been considered as preferredmodification sites.

A. Analytical Method of HC/LC Pairing

Novel HC/LC pairing analysis was devised in order to facilitate thesearch for the correct modification sites leading correct HC/LC pairing.

If the modification of proteins allows 100% discrimination of chains, alight chain LP (a positively charged light chain) is combined with aheavy chain HN (a negatively charged heavy chain), but not with HP (apositively charged heavy chain), to form a homologous HN/LP antibodiesconsisting of the 50 kD HN and 25 kD CP (positively charged common lightchains). It can be confirmed by the reduced SDS-PAGE gel. If themodification of proteins cannot distinguish the chains, the light chainLP will be combined with the heavy chains HP and HN, and will result inthe formation of three different Abs. It will form 60 kD HP as well as50 kD HN and 25 kD LP in the reduced SDS-PAGE gel.

B. Design for Symmetry and Asymmetry

Modification ws made in two different designs. The pattern of chargedistributions in the two Fab domains may be symmetrical or asymmetrical(see FIG. 19).

C. Variants pf HC/LC Pairing

Entire 21 hydrophobic residues were, separately or in combination,modified to lectrotatically interacting pair residues, and associated in21 sets of the variants (see Table 7).

( 

  7) Set HP HN LP LN Design Name Name HP (2B9) Name HN (4D9) Name LPName LN Symmetry V1 HPa F45K HNa F45D LPa F98K LNa F98D V2 HPb W47K HNbV47D LPb Y96K LNb Y96D V3 HPc W103K HNc W103D LPc P44K LNc P44D V4 HPdV37K HNd V37D LPa F98K LNa F98D V5 HPa F45K HNa F45D LPe Y87K LNe Y87DW1 HP1 L128K HN1 L128D LP1 F118K LN1 F118D W2 HP2 A141K HN2 A141D LP2F116K LN2 F116D W3 HP3 L145K HN3 L145D LP3 V133K LN3 V133D W4 HP4 V185KHN4 V185D LP4 L135K LN4 L135D W5 HP1 L128K HN1 L128D LP3 V133K LN3 V133DW6 HP2 A141K HN2 A141D LP4 L135K LN4 L135D W7 HP7 L145K/ HN7 L145D/ LP7V133K/ LN7 V133D/ V185K V185D L135K L135D W8 HP8 A141K/ HN8 A141D/ LP8F116K/ LN8 F116D/ V185K V185D L135K L135D Asymmetry V2P HPb W47K H0 — L0— LNb Y96D V3P HPc W103K H0 — L0 — LNc P44D W2P HP2 A141K H0 — L0 — LN2F116D W4P HP4 V185K H0 — L0 — LN4 L135D V3W4 HPc W103K HN4 V185D LP4L135K LNc P44D W4V3 HP4 V185K HNc W103D LPc P44K LN4 L135D V3V1 HPcW103K HNa F45D LPa F98K LNc P44D V3W1 HPc W103K HN1 L128D LP1 F118K LNcP44D

The degree of correct HC/LC pairing in the variant sets was measured bythe method of HC/LC pairing analysis, which was previously described.

SDS-PAGE Analysis

V3W1 set was chosen as the best variant leading to modified to correctHC/LC pairing among 21 sets (see FIG. 20).

1. A protein introduced an electrostatic interaction in a hydrophobicinteraction site wherein the electrostatic interaction is made between apositive charge and a negative charge, and the positive charge isintroduced by a modification from one hydrophobic amino acid to positivecharged material in a pair of amino acids in a site of hydrophobicinteraction selected from the sites of hydrophobic interaction ofprotein, and the negative charge is introduced by an modification fromthe other hydrophobic amino acid to negative charged material in thepair of amino acids.
 2. The protein introduced an electrostaticinteraction in a hydrophobic interaction site according to claim 1, ischaracterized by that the positive charged material is a basic aminoacid.
 3. The protein introduced an electrostatic interaction in ahydrophobic interaction site according to claim 1 characterized in thatthe negative charged material is an acidic amino acid.
 4. The proteinintroduced an electrostatic interaction in a hydrophobic interactionsite according to claim 1 characterized in that the positive chargedmaterial is a basic amino acid and the negative charged material is anacidic amino acid.
 5. The protein introduced an electrostaticinteraction in a hydrophobic interaction site according to claim 1characterized in that the hydrophobic amino acid is an amino acidselected from the groups comprising glycine, alanine, valine, leucine,isoleucine, methionine, proline, phenylalanine and tryptophan.
 6. Theprotein introduced an electrostatic interaction in a hydrophobicinteraction site according to claim 4 characterized in that the acidicamino acid is aspartic acid or glutamic acid, and the basic amino acidis an amino acid selected from the group comprising lysine, arginine andhistidine.
 7. An antibody introduced an electrostatic interaction in ahydrophobic interaction site wherein the electrostatic interaction ismade between a positive charge and a negative charge, and the positivecharge is introduced by a modification from one hydrophobic amino acidto positive charged material in a pair of amino acids in a site ofhydrophobic interaction selected from the sites of hydrophobicinteraction of antibody, and the negative charge is introduced by anmodification from the other hydrophobic amino acid to negative chargedmaterial in the pair of amino acids.
 8. The antibody introduced anelectrostatic interaction in a hydrophobic interaction site according toclaim 7 characterized in that the positive charged material is a basicamino acid.
 9. The antibody introduced an electrostatic interaction in ahydrophobic interaction site according to claim 7 characterized in thatthe negative charged material is an acidic amino acid.
 10. The antibodyintroduced an electrostatic interaction in a hydrophobic interactionsite according to claim 7 characterized in that the positive chargedmaterial is a basic amino acid and the negative charged material is anacidic amino acid.
 11. The antibody introduced an electrostaticinteraction in a hydrophobic interaction site according to claim 7characterized in that the hydrophobic amino acid is an amino acidselected from the groups comprising glycine, alanine, valine, leucine,isoleucine, methionine, proline, phenylalanine and tryptophan.
 12. Theantibody introduced an electrostatic interaction in a hydrophobicinteraction site according to claim 10 characterized in that the acidicamino acid is aspartic acid or glutamic acid, and the basic amino acidis an amino acid selected from the group comprising lysine, arginine andhistidine.
 13. A method of preparation of a protein with increased chainselectivity, the method being characterized in that the methodcomprising the following steps: (a) selecting a pair of amino acids in ahydrophobic interaction between polypeptide chain and polypeptide chain;(b) modifying one of hydrophobic amino acid to positive chargedmaterial, and the other hydrophobic amino acid to negative chargedmaterial in the selected pair of amino acids; and (c) binding withelectrostatic interaction by contacting between the positive chargedmaterial and the negative charged material.
 14. The method ofpreparation of a protein with increased chain selectivity according toclaim 13 characterized in that the positive charged material is a basicamino acid.
 15. The method of preparation of a protein with increasedchain selectivity according to claim 13 characterized in that thenegative charged material is an acidic amino acid.
 16. The method ofpreparation of a protein with increased chain selectivity according toclaim 13 characterized in that the positive charged material is a basicamino acid and the negative charged material is an acidic amino acid.17. The method of preparation of a protein with increased chainselectivity according to claim 13 characterized in that the hydrophobicamino acid is an amino acid selected from the groups comprising glycine,alanine, valine, leucine, isoleucine, methionine, proline, phenylalanineand tryptophan.
 18. The method of preparation of a protein withincreased chain selectivity according to claim 16 characterized in thatthe acidic amino acid is aspartic acid or glutamic acid, and the basicamino acid is an amino acid selected from the group comprising lysine,arginine and histidine.
 19. An antibody having binding force by anelectrostatic interaction between mutated amino acids wherein theelectrostatic interaction is made between the mutated amino acids in oneor more pairs of amino acids pair selected from the groups of pairs ofamino acids comprising 351 leucine and 351 leucine pair, 395 proline and397 valine pair, 395 proline and 395 proline pair, 407 tyrosine and 407tyrosine pair of hydrophobic interaction in CH3 domain; and 128 leucineand 118 phenylalanine pair, 128 leucine and 133 valine pair, 141 alanineand 116 phenylalanine pair, 141 alanine and 135 leucine pair, 145leucine and 133 valine pair, 170 phenylalanine and 135 leucine pair, 185valine and 118 phenylalanine pair and 185 valine and 135 leucine pairbetween CH1 domain and CL domain of human antibody, and the mutatedamino acids is made by mutating one amino acid in the selected aminoacid pair to acidic amino acid selected from aspartic acid or glutamicacid, and by mutating the other amino acid of the selected amino acidpair to basic amino acid selected from the group of amino acidscomprising lysine, arginine and histidine.
 20. The antibody according toclaim 19 characterized in that the mutated amino acid pair is selectedfrom the group of set comprising the combination of Z0 to Z14 of Table4.
 21. The antibody according to claim 19 wherein the antibody is afusion antibody by combination a pair of ectodomain selected from thegroup comprising TNR2, Her3, Tie2, TGFbR1, BMPbR1, Il-12R-b1, IL-4Ra,ITGA4, ITGA2B, INFAR1, IL-12A, IL-4, InFa, BMP2, IL-1R1L, IL-17RA,IL-17A, Fas, FltD2, Her1, Tie1,TGFbR2, IL-12R-b2, IL-13Ra1, ITGB1,ITGB3, INFAR2, IL-12B, IL-13, INFb, BMP7, IL-1RAP, IL-17RC and IL-17F.22. The antibody according to claim 21 wherein the antibody is a fusionantibody with a combination of ectodomain selected from the groupcomprising Her2/FltD2 combination, Her1/Her3 combination and Tie/Tie2combination.
 23. The antibody according to claim 19 wherein the antibodyis an bispecific antibody with heavy chains mutated with the combinationof Z14, and common light chains comprising 4D9 ectodomain for A-typeinfluenza virus and 2B9 ectodomain for B-type influenza virus.
 24. Amethod of measuring coupling extent of heavy chain and light chain of abispecific antibody by using the antibody according to claim
 23. 25. Anantibody increased the coupling extent between heavy chain and lightchain consisting of a combination selected from the group of combinationof V1-V5, W1-W8, V2p, V3p, W4p, V3W4, W4v3, V3v1 of heavy positive chain(HP), heavy negative chain (HN), light positive chain (LP) and lightnegative chain (LN) of Table
 7. 26. An antibody increased the couplingextent between heavy chain and light chain according to claim 25,wherein 103 tryptophan in one of heavy chain is mutated to lysine; 128lysine in the other heavy chain is mutated to aspartic acid; 118phenylalanine in one of light chain is mutated to lysine; and 44 prolinein other light chain is mutated to aspartic acid.